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Biological effect of intermittent radiation exposure in vivo: Recovery from sublethal damage versus reoxygenation
Radiotherapy and Oncology 86 (2008) 369–374 www.thegreenjournal.com
DNA repair
Biological effect of intermittent radiation exposure in vivo: Recovery from sublethal damage versus reoxygenation Natsuo Tomita*, Yuta Shibamoto, Masato Ito, Hiroyuki Ogino, Chikao Sugie, Shiho Ayakawa, Hiromitsu Iwata Department of Radiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Abstract Purpose: In vivo effects of intermittent irradiation are influenced by recovery from sublethal damage (SLDR) and reoxygenation, so contribution of the two factors were investigated using murine tumors. Methods and materials: 1-cm-diameter SCCVII tumors growing in the legs of C3H/HeN mice were used. First, effects of 5 fractions of 6 Gy given at intervals of 2.5–15 min were compared using an in vivo–in vitro assay, by clamping the tumor-bearing legs to exclude the influence of reoxygenation. In the second and third experiments, changes in the hypoxic fraction at 0–15 min after 13 or 5 Gy were assessed by a paired cell survival method. Fourth, effects of 5 fractions of 5 Gy given at intervals of 3–10 min under conditions of limited reoxygenation were compared using a growth delay assay. Results: Cell survival from clamped tumors tended to increase with elongation of the intervals, but not significantly. The hypoxic fraction tended to decrease at 5–15 min from the level immediately after irradiation. Effects on tumor growth tended to decrease with elongation of the intervals. Conclusions: Reoxygenation occurring within 5–15 min appeared to compensate for SLDR in SCCVII tumors. When reoxygenation was limited, the decrease of radiation effect occurred due to SLDR. c 2007 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 86 (2008) 369–374.
Keywords: Recovery from sublethal damage; In vivo–in vitro assay; Reoxygenation; Stereotactic irradiation; Stereotactic radiotherapy
Recently, stereotactic radiotherapy (SRT) has been increasingly used in the clinic and has shown clinical benefit in patients with malignancy [9,23]. This new technique generally employs multiple arc or fixed beams from a linear accelerator (linac), and therefore a considerable time is required for setup for delivering each beam, including positioning of the gantry and couch. Since moderate doses (usually 2–4 Gy) are given at several-minute intervals in linac SRT, the total time required for one treatment generally ranges from 15 to 60 min. In such a treatment, there is a concern that the effect of radiation may be reduced during intermittent exposures due to recovery from sublethal damage (SLDR) that Elkind and coworkers reported in the 1960s [6–8]. In an in vitro study from our laboratory in which total doses of 2 and 8 Gy were delivered in 2, 5 or 10 fractions, the biological effects of radiation significantly decreased when interfraction intervals were 2 min or longer in EMT6 and SCCVII cells [17]. The dose-modifying factor appeared to be 1.08–1.16 when the total time for irradiation was between 20 and 30 min. In the subsequent in vitro study investigating various fractionation schedules, the decrease of radiation effect with prolongation of total radiation time was also confirmed [16]. On the other hand, results of our
previous in vivo study were in striking contrast with those of the in vitro studies [21]. In the 2-fraction experiments, the increase in cell survival with elongation of intervals was much less than that observed in the in vitro study. Furthermore, the relative survival significantly decreased when the interruption between fractions was 15 min in SCCVII cells. In both of the 5- and 10-fraction experiments, no significant increase in cell survival resulting from fractionation was observed. Moreover, cell survival decreased at various time points by interruption of radiation. According to Fowler et al. [10], dose-modifying factors of 1.1–1.35 would be expected due to intermittent 20 Gy irradiation taking 0.5–1 h in tissues with an a/b ratio of 3 Gy and 50:50 repair half-times of 0.2 0.4 h + 4.0 h. These parameters almost agree with those of the cell lines used in our previous in vivo study. From these conflicting in vitro and in vivo results, we considered that SLDR in vivo might be counterbalanced by other phenomena such as reoxygenation that sensitizes tumor cells to subsequent irradiation. To verify this hypothesis, we investigated in the present study the effect of intermittent radiation under conditions of restricted reoxygenation as well as the magnitude of reoxygenation during 2.5–15 min of radiation interruption.
0167-8140/$ - see front matter c 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2007.08.007
370
Biological effect of intermittent radiation
Materials and methods Cell line and mice SCCVII cells were cultured in Eagle’s minimum essential medium containing 12.5% fetal bovine serum. Characteristics of this cell line have been described previously [18]. On dose-survival curves under aerobic conditions, SCCVII had an a/b ratio of 1.7 Gy (Y. Shibamoto, unpublished data, 2003). The cells subcultured on the day before transplantation were inoculated subcutaneously (200,000 cells per leg) into both hind legs (for in vivo–in vitro assay) or the right hind legs (for growth delay assay) of female 8-week-old C3H/HeN mice. Experiments were performed when the tumor reached approximately 1 cm in diameter 10 days later. This experiment using the tumors of this size was approved by an Institutional Ethics Committee for animal experiments.
Irradiation Irradiation was performed using a 210-kV X-ray machine (10 mA with a 2-mm Al filter; Chubu Medical Co., Matsusaka, Japan) at a dose rate of 2 Gy/min. In the first to third experiments, mice received whole-body irradiation without anesthesia or physical restraint. In the fourth experiment, tumor-bearing mice were immobilized in a jig with their legs fixed using adhesive tapes to deliver focal irradiation. Mice were kept under these conditions until all irradiation finished. The dose was calibrated using a RAMTEC 1000 dosimeter (Toyo Medic, Tokyo, Japan).
Radiation schedule First, 5 fractions of 6 Gy were given at an interval of 0, 2.5, 5, 7.5, 10 or 15 min by clamping the tumor-bearing legs during radiation. During intervals between fractions, the leg clamping was released. Single doses of 24 and 27 Gy were also given without interruption to estimate dose-modifying factors. This experiment was designed to investigate the change of radiation effect excluding the influence of reoxygenation in vivo, following the experimental protocol of our previous in vivo study [21]. In the second and third experiments, tumor-bearing mice were irradiated to the whole body with a priming dose of 13 Gy and 5 Gy, respectively. A dose of 13 Gy was used to kill nearly all oxygenated cells in the tumors, and a 5 Gy dose was used to leave some oxygenated cells alive [11,20]. At various intervals (0, 2.5, 5, 7.5, 10 and 15 min) thereafter, a second dose of 15 Gy was given to the mice, with half of them killed by cervical dislocation and the remaining ones kept alive, to determine the change in the hypoxic fraction by an in vivo–in vitro assay. Sequences of irradiation to each group were changed every time to avoid any possible biases. Fourth, tumor-bearing mice were irradiated with a single dose of 20 or 25 Gy, or 5 fractions of 5 Gy with interfraction intervals of 3, 6 or 10 min.
were then counted and plated onto culture dishes in triplicate in the above-mentioned minimum essential medium. The mean tumor weight (±SD) was 0.36 ± 0.08, 0.41 ± 0.06 and 0.39 ± 0.09 g in the first, second and third experiments, respectively. There were no significant differences in mean tumor weight among the groups in each experiment. After 9 days, colonies were fixed with 75% ethanol and stained with Giemsa. The control plating efficacy (±SD) was 16 ± 9%, 17 ± 4% and 14 ± 6% in the first, second and third experiments, respectively. The surviving fractions for irradiated groups were normalized by the plating efficiency. In the first experiment, the surviving fraction for the control groups receiving 30 Gy continuously was regarded as 1 in each experiment and relative survival was calculated for the intermittent radiation groups. Similarly in the second and third experiments, the surviving fractions for the tumors receiving second irradiation in dead mice at an interval of 0 min were regarded as 1 and relative survival was calculated for the other groups. To use relative survival instead of actual surviving fractions was considered appropriate, since some experimental points were lost because of bacterial contamination especially in the first experiment. Therefore, relative survival was calculated in each experiment, and it was averaged thereafter. The high rate of contamination was considered to be due to the effect of clamping that caused tumor bleeding. Two groups were assigned for the control in every experiment, and the average of the two was used to calculate the relative surviving fractions in the other groups. The mean relative surviving fraction was calculated from 6 to 8 determinations on average in all experiments. Differences in relative surviving fractions between pairs of groups were examined by Student’s t-test. In the fourth experiment, 18 tumors from 18 mice were used for each group. To assess tumor growth delay, the 3 dimensions of each tumor were measured every other day with calipers, and the tumor volume was estimated using the formula p/6 · product of the 3 dimensions. Tumor growth time was defined as the time required after the first day of treatment for a tumor to reach 1.5 times the initial volume. Differences between pairs of tumor growth curves were examined by analysis of covariance.
Measurement of the hypoxic fraction The hypoxic fraction was determined from the ratio of surviving fractions of tumor cells irradiated in air-breathing mice to that irradiated in dead mice [12,14]. To determine the hypoxic fraction of unirradiated 1 cm SCCVII tumors, a single dose of 28 Gy was delivered to tumors in air-breathing and dead mice.
Results Assay In the first to third experiments, cell survival was determined by an in vivo–in vitro assay. Four tumors from 2 mice were used for each point. Tumors were excised immediately after radiation, weighed, minced with scissors, and treated with 0.1% neutral protease solution for 30 min. Tumor cells
Fig. 1 shows the results of the first experiment in which 5 fractions of 6 Gy were given to the mice with their tumorbearing legs clamped during irradiation. The average surviving fraction after 24, 27 and 30 Gy doses given continuously was 0.013 (CI: 0.0070–0.026), 0.0073 (CI: 0.0018–0.030) and 0.0022 (CI: 0.0010–0.0049), respectively. Cell survival
N. Tomita et al. / Radiotherapy and Oncology 86 (2008) 369–374
*
4
2
*
Relative surviving fraction
Relative surviving fraction
30 Gy 3
2
1
0
2.5
5
7.5
10
15
Interval (min)
27
1
0.5
0
24
Dose (Gy)
Fig. 1. Relative surviving fractions of SCCVII tumor cells after 30 Gy given in 5 fractions of 6 Gy at intervals of 0–15 min and after 24 and 27 Gy as a single dose. Surviving fraction after continuous 30 Gy irradiation was regarded as 1. Bars represent SE of 6 determinations on average. *p < 0.05 against the control 30 Gy (0 min) group.
tended to increase with elongation of the interfraction interval, but not significantly. The hypoxic fraction for the control 1 cm SCCVII tumors was 18% (CI: 14–23%). Fig. 2 shows the results of the second experiment investigating reoxygenation after a priming dose of 13 Gy. The surviving fraction of the groups given second irradiation after cervical dislocation at intervals of 0 min was 0.00070 (CI: 0.00045–0.00094). No reoxygenation was seen when the interval was 0 or 2.5 min (hypoxic fraction: 100% at both intervals), but when the interruption was 5 min or longer, the hypoxic fraction was approximately 70% (67% at 5 min, 76% at 10 min and 66% at 15 min). The relative surviving fractions of the two groups were significantly different at 5 min after the priming dose 2.5
Relative surviving fraction
1.5
0 -2.5 0
2 1.5 1
2.5
5
7.5 10 Interval (min)
12.5
15
17.5
Fig. 3. Relative surviving fractions of SCCVII tumor cells after a priming dose of 5 Gy and a second dose of 15 Gy given at 0- to 15min intervals to air-breathing (s — s) or dead (d — d) mice. The surviving fraction of the group killed immediately after the priming irradiation was regarded as 1. Bars represent SE of 8 determinations on average.
(p = 0.047), indicating occurrence of significant reoxygenation. Fig. 3 shows the results of the third experiment investigating reoxygenation after a priming dose of 5 Gy. The surviving fraction of the groups given second irradiation after cervical dislocation at intervals of 0 min was 0.0081 (CI: 0.0038–0.012). Almost no reoxygenation appeared to occur within 5 min (hypoxic fraction: 83% at 0 min, 77% at 2.5 min and 81% at 5 min), but the hypoxic fraction tended to decrease to 57% (CI: 19–95%) at 10 min and 56% (CI: 31– 82%) at 15 min. Table 1 summarizes changes of hypoxic fractions at various intervals in the second and third experiments. Fig. 4 shows the results of the growth delay experiment. The effect of 25 Gy given in 5 fractions at 3-, 6- or 10-min interfraction intervals was significantly lower than that of a single 25 Gy dose (25 Gy as a single dose versus 25 Gy given in 5 fractions at 3-, 6- and 10-min intervals: p = 0.037, <0.0001, and <0.0001, respectively). The effect decreased or tended to decrease with elongation of the interfraction intervals (3-min intervals versus 6-min intervals, p = 0.045; 3-min intervals versus 10-min intervals, p = 0.002; 6-min
Table 1 Changes in the hypoxic fraction of SCC tumor cells at various intervals (0–15 min) after a priming dose of 13 or 5 Gy
0.5
Interval (min)
0 -2.5
371
0
2.5
5
7.5
10
12.5
15
17.5
Interval (min) Fig. 2. Relative surviving fractions of SCCVII tumor cells after a priming dose of 13 Gy and a second dose of 15 Gy given at 0- to 15min intervals to air-breathing (s — s) or dead (d — d) mice. The surviving fraction of the group killed immediately after the priming irradiation was regarded as 1. Bars represent SE of 8 determinations on average.
0 2.5 5 10 15
Hypoxic fraction (%) After 13 Gy
After 5 Gy
100 (61–100) 100 (64–100) 67 (41–93) 76 (34–100) 66 (37–100)
83 77 81 57 56
(68–99) (46–100) (52–100) (19–95) (31–82)
Figures in parenthesis are 95% confidence intervals (CI). The control SCCVII tumors had a hypoxic fraction of 18% (CI: 14– 23%).
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Biological effect of intermittent radiation
Relative tumor volume (%)
200
150
100
50
0 -10
0
10
20
30
40
50
60
Days after irradiation Fig. 4. Relative tumor volumes of SCCVII tumor cells after 20 or 25 Gy as a single dose, or 5 fractions of 5 Gy given at interfraction intervals of 3, 6 or 10 min. Tumor volumes before irradiation for each group were regarded as 100%. Bars represent SE of 18 mice on average. n, control; m, 20 Gy as a single dose; d, 25 Gy as a single dose; h, 25 Gy in 5 fractions given at 3-min intervals; j, 25 Gy in 5 fractions at 6-min intervals; s, 25 Gy in 5 fractions at 10-min intervals. Bars represent SE of 18 mice on average (for the irradiated groups).
Table 2 Tumor growth time (TGT) to reach 1.5 times the initial volume Radiation schedule
Control 20 Gy as a single dose 25 Gy as a single dose 25 Gy in 5 fractions at 3-min intervals 25 Gy in 5 fractions at 6-min intervals 25 Gy in 5 fractions at 10-min intervals
TGT (days) Mean
SE
3.2 39.5 55.1 52.4 50.5 46.9
1.4 5.1 3.1 3.9 3.8 4.6
intervals versus 10-min intervals, p = 0.24). Tumor growth time for each group is shown in Table 2.
Discussion In a previous study conducted by Shibamoto et al. [19] using 1-cm-diameter SCCVII tumors from the same frozen stock as used in this study, the hypoxic fraction was 5.4% (95% confidence interval [CI]: 4.0–7.4%) in air-breathing mice irradiated without anesthesia or physical restraint, but under the conditions for the growth delay assay (conditions for the fourth experiment in this study), the hypoxic fraction increased to 28% (CI: 20–40%). In other 3 studies by Shibamoto’s groups [11,18,20], the hypoxic fractions of the 1-cm-diameter SCCVII tumors from the same stock were 8.5% (CI: 6.9–11%), 10% (CI: 8–13%) and 9.1% (6.9–12%), respectively. Since immobilizing mice in jigs and taping their legs artificially increases the hypoxic fraction, oxygenation status is considered to remain poorer when tumor-
bearing mice are kept under these conditions [4,19]; in the fourth experiment (growth delay assay), reoxygenation was thus considered to be restricted compared with that in tumors in air-breathing mice with no physical restraint. Also from these observations, it was considered that mice had to be irradiated without physical restraint or anesthesia to investigate reoxygenation in the second and third experiments. The present study was undertaken in an attempt to find explanation for the discrepancy seen between our previous in vitro and in vivo studies; significant decreases in radiation effects were observed by posing intervals between fractions in vitro but they were not observed in vivo [16,17,21]. The apparent increase in cell survival in vitro should be due to SLDR, so questions arose as to whether SLDR might not occur in vivo or SLDR might be counterbalanced by other phenomena that sensitize tumor cells to subsequent irradiation. The first experiment was undertaken to investigate SLDR in vivo by excluding reoxygenation of tumor cells during fractionated irradiation. As a result, tumor cell survival tended to increase with elongation of intervals, but statistically, the increases were not significant. So, SLDR in vivo could not be proven with this experiment. One of the reasons why no statistically significant increases could be obtained in the first experiment may be the relatively large experimental errors associated with in vivo– in vitro studies. Moreover, clamping of tumor-bearing legs caused tumor bleedings, which led to a high frequency of bacterial contamination in vitro so that many experimental points were lost. This is also a reason why we used relative survival for analysis. This experiment was repeated 5 times (each time making 2 determinations per time point), so we did not further attempt to obtain statistical significance. The second and third experiments investigated the magnitude of reoxygenation during 2.5–15 min of radiation interruption. Several possible mechanisms for reoxygenation have been proposed, but rapid reoxygenation, if any, should mainly be due to restoration of blood flow to acutely hypoxic cells [1,13]. Kitakabu et al. [11] argued that the pattern of reoxygenation after a single 13 Gy dose seemed to be biphasic, comprising an extremely rapid phase within 1 h and a comparatively slow phase during the following 12– 72 h in SCCVII tumors. Reoxygenation within 1 h has also been reported in RIF-1 tumors [5,15], although it was hardly observed in EMT6 tumors [15,22]. Evidences showing that changes in blood perfusion can occur in subcutaneous SCCVII tumors within a 20-min interval were found in other studies [2,3]. Therefore, reoxygenation may occur even within 20 min in vivo. However, magnitude of reoxygenation within such a short time has not been well studied, so we attempted to clarify it in SCCVII tumors. In the present study, apparent reoxygenation was observed at 5 min after a priming dose of 13 Gy. Trends towards reoxygenation were also observed at 10 and 15 min, although the CI included 100%. After a priming dose of 5 Gy, both oxygenated and hypoxic cells can survive, but the hypoxic fraction tended to be lower at 10 and 15 min than at 0, 2.5 and 5 min. Taking these results together, reoxygenation appeared to take place within 15 min in SCCVII tumors. Then a question remains as to whether reoxygenation of this magnitude can counterbalance SLDR
N. Tomita et al. / Radiotherapy and Oncology 86 (2008) 369–374
in vivo. In the second experiment, the hypoxic fraction was 100% at 0- and 2.5-min intervals, while it was 67% at 5 min interval, so approximately one-third of tumor cells reoxygenated within 5 min after 13 Gy. Surviving fractions for tumors irradiated in dead mice tended to increase with elongation of the intervals due to SLDR, but those for tumors irradiated in living mice remained at a level similar to that at 0 min, after 5-min or longer intervals following 13 Gy. In the third experiment, the hypoxic fraction was about 80% at 0- to 5-min intervals and about 55% at 10and 15-min intervals, so approximately one-third of tumor cells might also have reoxygenated at 10–15 min after 5 Gy. Surviving fractions for tumors irradiated in dead mice also tended to increase with elongation of the intervals due to SLDR, but those for tumors irradiated in living mice returned to a level similar to that at 0 min after 10- and 15min intervals. These result would indicate that reoxygenation of about one-third of hypoxic tumor cells would counterbalance SLDR in vivo. In the fourth experiment, the fractionated groups had faster tumor regrowth than the continuously-irradiated control group, and the effect of radiation tended to decrease with elongation of interfraction intervals. Thus, SLDR appeared to have occurred in this experiment, since no other explanations seemed relevant. As stated above, the hypoxic fraction in SCCVII tumors artificially increased from 5.4% to 28% by taping tumor-bearing legs [19]. In the present study, the control hypoxic fraction was 18% so that the hypoxic fraction of the taped and immobilized mice might have been somewhat higher. Anyway, the effects of intermittent exposure appeared to decrease, as compared with continuous irradiation, due to SLDR under these conditions. Results of the present study are summarized as follows. Two phenomena have been demonstrated in these tumor models that effect response in opposite ways during fractionated radiotherapy. The first was increased reoxygenation that can sensitize tumors (second and third experiments), and the second was presumably increased recovery (fourth experiment), although this was not formally proven in the first experiment. The reason why we could not prove SLDR in the first experiment was considered to be due to technical problems associated with leg clamping. In the fourth experiment, however, SLDR in vivo was considered to be proven by using the experimental setup which artificially restricted oxygenation. Potential relevance of our four series of experiments (Refs. [16,17,21] and the present study) for clinical radiation therapy may be summarized as follows. SLDR is a significant factor decreasing the effect of intermittent irradiation given over 20 min or longer. In some tumors in vivo, the decrease in effect may be compensated by rapid reoxygenation. However, the magnitude and velocity of reoxygenation in human tumors are almost unknown, so it cannot be concluded that the clinical effect of intermittent irradiation as employed in linac SRT is nearly equal to that of continuous irradiation. Biologically, investigations using tumors that are closer to human tumors are desirable. Clinically, the time for irradiation may better be kept as short as possible, considering the possibility of slow or insufficient reoxygenation of the tumor.
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Conflict of interest statement No conflicts of interest exist for any of the authors.
Acknowledgement This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Corresponding author. Natsuo Tomita, Department of Radiology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail address: [email protected] Received 25 March 2007; received in revised form 18 August 2007; accepted 20 August 2007; Available online 18 September 2007
References [1] Brown JM. Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation. Br J Radiol 1979;52:650–6. [2] Chaplin DJ, Durand RE, Olive PL. Acute hypoxia in tumors: implications for modifiers of radiation effects. Int J Radiat Oncol Biol Phys 1986;12:1279–82. [3] Chaplin DJ, Olive PL, Durand RE. Intermittent blood flow in a murine tumor: radiobiological effects. Cancer Res 1987;37:597–601. [4] Cullen BM, Walker HC. The effect of several different anesthetics on the blood pressure and heart rate of the mouse and on the radiation response of the mouse sarcoma RIF-1. Int J Radiat Biol 1985;48:761–71. [5] Dorie MJ, Kallman RF. Reoxygenation in the RIF-1 tumor. Int J Radiat Oncol Biol Phys 1984;10:687–93. [6] Elkind MM, Sutton H. Radiation response of mammalian cells grown in culture. I. Repair of X-ray damage in surviving Chinese hamster cells. Radiat Res 1960;13:556–93. [7] Elkind MM, Alescio T, Swain RW, Moses WB, Sutton H. Recovery of hypoxic mammalian cells from sub-lethal X-ray damage. Nature 1964;202:1190–3. [8] Elkind MM. Cell-cycle sensitivity, recovery from radiation damage and a new paradigm for risk assessment. Int J Radiat Biol 1997;71:657–65. [9] Ernst-Stecken A, Papiez L, Ganslandt O, et al. Phase II trial of hypofractionated stereotactic radiotherapy for brain metastases: results and toxicity. Radiother Oncol 2006;81:18–24. [10] Fowler JF, Welsh JS, Howard SP. Loss of biological effect in prolonged fraction delivery. Int J Radiat Oncol Biol Phys 2004;59:242–9. [11] Kitakabu Y, Shibamoto Y, Sasai K, Ono K, Abe M. Variations of the hypoxic fraction in the SCCVII tumors after single dose and during fractionated radiation therapy: assessment without anesthesia or physical restraint of mice. Int J Radiat Oncol Biol Phys 1991;20:709–14. [12] Kim IH, Brown JM. Reoxygenation and rehypoxiation in the SCCVII mouse tumor. Int J Radiat Oncol Biol Phys 1994;29:493–7. [13] Kallman RF. The phenomenon of reoxygenation and its implications for fractionated radiotherapy. Radiology 1972;105:135–42. [14] Moulder JE, Rockwell S. Hypoxic fractions of solid tumors: experimental techniques, methods of analysis, and a survey of existing data. Int J Radiat Oncol Biol Phys 1984;10:695–712. [15] Murata R, Shibamoto Y, Sasai K, et al. Reoxygenation after single irradiation in rodent tumors of different types and sizes. Int J Radiat Oncol Biol Phys 1996;34:859–65.
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[16] Ogino H, Shibamoto Y, Sugie C, Ito M. Biological effects of intermittent radiation in cultured tumor cells: influence of fraction number and dose per fraction. J Radiat Res 2005;46:401–6. [17] Shibamoto Y, Ito M, Sugie C, Ogino H, Hara M. Recovery from sublethal damage during intermittent exposures in cultured tumor cells: implications for dose modification in radiosurgery and IMRT. Int J Radiat Oncol Biol Phys 2004;59:1484–90. [18] Shibamoto Y, Yukawa Y, Tsutsui K, Takahashi M, Abe M. Variation in the hypoxic fraction among mouse tumors of different types, sizes, and sites. Jpn J Cancer Res 1986;77:908–15. [19] Shibamoto Y, Sasai K, Abe M. The radiation response of SCCVII tumor cells in C3H/He mice varies with the irradiation conditions. Radiat Res 1987;109:352–4.
[20] Shibamoto Y, Kitakabu Y, Murata R, et al. Reoxygenation in the SCCVII tumor after KU-2285 sensitization plus single or fractionated irradiation. Int J Radiat Oncol Biol Phys 1994;29:583–6. [21] Sugie C, Shibamoto Y, Ito M, et al. Radiobiologic effect of intermittent radiation exposure in murine tumors. Int J Radiat Oncol Biol Phys 2006;64:619–24. [22] Shibamoto Y, Nishimura Y, Nishidai T, Takahasi M, Abe M. Reoxygenation after a single dose of 15 Gy in the EMT6/KU sarcoma. Nippon Igaku Hoshasen Gakkai Zasshi 1986;46:1319–23 [in Japanese]. [23] Wersa ¨ll PJ, Blomgren H, Lax I, et al. Extracranial stereotactic radiotherapy for primary and metastatic renal cell cell carcinoma. Radiother Oncol 2005;77:88–95.
DNA repair
Biological effect of intermittent radiation exposure in vivo: Recovery from sublethal damage versus reoxygenation Natsuo Tomita*, Yuta Shibamoto, Masato Ito, Hiroyuki Ogino, Chikao Sugie, Shiho Ayakawa, Hiromitsu Iwata Department of Radiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Abstract Purpose: In vivo effects of intermittent irradiation are influenced by recovery from sublethal damage (SLDR) and reoxygenation, so contribution of the two factors were investigated using murine tumors. Methods and materials: 1-cm-diameter SCCVII tumors growing in the legs of C3H/HeN mice were used. First, effects of 5 fractions of 6 Gy given at intervals of 2.5–15 min were compared using an in vivo–in vitro assay, by clamping the tumor-bearing legs to exclude the influence of reoxygenation. In the second and third experiments, changes in the hypoxic fraction at 0–15 min after 13 or 5 Gy were assessed by a paired cell survival method. Fourth, effects of 5 fractions of 5 Gy given at intervals of 3–10 min under conditions of limited reoxygenation were compared using a growth delay assay. Results: Cell survival from clamped tumors tended to increase with elongation of the intervals, but not significantly. The hypoxic fraction tended to decrease at 5–15 min from the level immediately after irradiation. Effects on tumor growth tended to decrease with elongation of the intervals. Conclusions: Reoxygenation occurring within 5–15 min appeared to compensate for SLDR in SCCVII tumors. When reoxygenation was limited, the decrease of radiation effect occurred due to SLDR. c 2007 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 86 (2008) 369–374.
Keywords: Recovery from sublethal damage; In vivo–in vitro assay; Reoxygenation; Stereotactic irradiation; Stereotactic radiotherapy
Recently, stereotactic radiotherapy (SRT) has been increasingly used in the clinic and has shown clinical benefit in patients with malignancy [9,23]. This new technique generally employs multiple arc or fixed beams from a linear accelerator (linac), and therefore a considerable time is required for setup for delivering each beam, including positioning of the gantry and couch. Since moderate doses (usually 2–4 Gy) are given at several-minute intervals in linac SRT, the total time required for one treatment generally ranges from 15 to 60 min. In such a treatment, there is a concern that the effect of radiation may be reduced during intermittent exposures due to recovery from sublethal damage (SLDR) that Elkind and coworkers reported in the 1960s [6–8]. In an in vitro study from our laboratory in which total doses of 2 and 8 Gy were delivered in 2, 5 or 10 fractions, the biological effects of radiation significantly decreased when interfraction intervals were 2 min or longer in EMT6 and SCCVII cells [17]. The dose-modifying factor appeared to be 1.08–1.16 when the total time for irradiation was between 20 and 30 min. In the subsequent in vitro study investigating various fractionation schedules, the decrease of radiation effect with prolongation of total radiation time was also confirmed [16]. On the other hand, results of our
previous in vivo study were in striking contrast with those of the in vitro studies [21]. In the 2-fraction experiments, the increase in cell survival with elongation of intervals was much less than that observed in the in vitro study. Furthermore, the relative survival significantly decreased when the interruption between fractions was 15 min in SCCVII cells. In both of the 5- and 10-fraction experiments, no significant increase in cell survival resulting from fractionation was observed. Moreover, cell survival decreased at various time points by interruption of radiation. According to Fowler et al. [10], dose-modifying factors of 1.1–1.35 would be expected due to intermittent 20 Gy irradiation taking 0.5–1 h in tissues with an a/b ratio of 3 Gy and 50:50 repair half-times of 0.2 0.4 h + 4.0 h. These parameters almost agree with those of the cell lines used in our previous in vivo study. From these conflicting in vitro and in vivo results, we considered that SLDR in vivo might be counterbalanced by other phenomena such as reoxygenation that sensitizes tumor cells to subsequent irradiation. To verify this hypothesis, we investigated in the present study the effect of intermittent radiation under conditions of restricted reoxygenation as well as the magnitude of reoxygenation during 2.5–15 min of radiation interruption.
0167-8140/$ - see front matter c 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2007.08.007
370
Biological effect of intermittent radiation
Materials and methods Cell line and mice SCCVII cells were cultured in Eagle’s minimum essential medium containing 12.5% fetal bovine serum. Characteristics of this cell line have been described previously [18]. On dose-survival curves under aerobic conditions, SCCVII had an a/b ratio of 1.7 Gy (Y. Shibamoto, unpublished data, 2003). The cells subcultured on the day before transplantation were inoculated subcutaneously (200,000 cells per leg) into both hind legs (for in vivo–in vitro assay) or the right hind legs (for growth delay assay) of female 8-week-old C3H/HeN mice. Experiments were performed when the tumor reached approximately 1 cm in diameter 10 days later. This experiment using the tumors of this size was approved by an Institutional Ethics Committee for animal experiments.
Irradiation Irradiation was performed using a 210-kV X-ray machine (10 mA with a 2-mm Al filter; Chubu Medical Co., Matsusaka, Japan) at a dose rate of 2 Gy/min. In the first to third experiments, mice received whole-body irradiation without anesthesia or physical restraint. In the fourth experiment, tumor-bearing mice were immobilized in a jig with their legs fixed using adhesive tapes to deliver focal irradiation. Mice were kept under these conditions until all irradiation finished. The dose was calibrated using a RAMTEC 1000 dosimeter (Toyo Medic, Tokyo, Japan).
Radiation schedule First, 5 fractions of 6 Gy were given at an interval of 0, 2.5, 5, 7.5, 10 or 15 min by clamping the tumor-bearing legs during radiation. During intervals between fractions, the leg clamping was released. Single doses of 24 and 27 Gy were also given without interruption to estimate dose-modifying factors. This experiment was designed to investigate the change of radiation effect excluding the influence of reoxygenation in vivo, following the experimental protocol of our previous in vivo study [21]. In the second and third experiments, tumor-bearing mice were irradiated to the whole body with a priming dose of 13 Gy and 5 Gy, respectively. A dose of 13 Gy was used to kill nearly all oxygenated cells in the tumors, and a 5 Gy dose was used to leave some oxygenated cells alive [11,20]. At various intervals (0, 2.5, 5, 7.5, 10 and 15 min) thereafter, a second dose of 15 Gy was given to the mice, with half of them killed by cervical dislocation and the remaining ones kept alive, to determine the change in the hypoxic fraction by an in vivo–in vitro assay. Sequences of irradiation to each group were changed every time to avoid any possible biases. Fourth, tumor-bearing mice were irradiated with a single dose of 20 or 25 Gy, or 5 fractions of 5 Gy with interfraction intervals of 3, 6 or 10 min.
were then counted and plated onto culture dishes in triplicate in the above-mentioned minimum essential medium. The mean tumor weight (±SD) was 0.36 ± 0.08, 0.41 ± 0.06 and 0.39 ± 0.09 g in the first, second and third experiments, respectively. There were no significant differences in mean tumor weight among the groups in each experiment. After 9 days, colonies were fixed with 75% ethanol and stained with Giemsa. The control plating efficacy (±SD) was 16 ± 9%, 17 ± 4% and 14 ± 6% in the first, second and third experiments, respectively. The surviving fractions for irradiated groups were normalized by the plating efficiency. In the first experiment, the surviving fraction for the control groups receiving 30 Gy continuously was regarded as 1 in each experiment and relative survival was calculated for the intermittent radiation groups. Similarly in the second and third experiments, the surviving fractions for the tumors receiving second irradiation in dead mice at an interval of 0 min were regarded as 1 and relative survival was calculated for the other groups. To use relative survival instead of actual surviving fractions was considered appropriate, since some experimental points were lost because of bacterial contamination especially in the first experiment. Therefore, relative survival was calculated in each experiment, and it was averaged thereafter. The high rate of contamination was considered to be due to the effect of clamping that caused tumor bleeding. Two groups were assigned for the control in every experiment, and the average of the two was used to calculate the relative surviving fractions in the other groups. The mean relative surviving fraction was calculated from 6 to 8 determinations on average in all experiments. Differences in relative surviving fractions between pairs of groups were examined by Student’s t-test. In the fourth experiment, 18 tumors from 18 mice were used for each group. To assess tumor growth delay, the 3 dimensions of each tumor were measured every other day with calipers, and the tumor volume was estimated using the formula p/6 · product of the 3 dimensions. Tumor growth time was defined as the time required after the first day of treatment for a tumor to reach 1.5 times the initial volume. Differences between pairs of tumor growth curves were examined by analysis of covariance.
Measurement of the hypoxic fraction The hypoxic fraction was determined from the ratio of surviving fractions of tumor cells irradiated in air-breathing mice to that irradiated in dead mice [12,14]. To determine the hypoxic fraction of unirradiated 1 cm SCCVII tumors, a single dose of 28 Gy was delivered to tumors in air-breathing and dead mice.
Results Assay In the first to third experiments, cell survival was determined by an in vivo–in vitro assay. Four tumors from 2 mice were used for each point. Tumors were excised immediately after radiation, weighed, minced with scissors, and treated with 0.1% neutral protease solution for 30 min. Tumor cells
Fig. 1 shows the results of the first experiment in which 5 fractions of 6 Gy were given to the mice with their tumorbearing legs clamped during irradiation. The average surviving fraction after 24, 27 and 30 Gy doses given continuously was 0.013 (CI: 0.0070–0.026), 0.0073 (CI: 0.0018–0.030) and 0.0022 (CI: 0.0010–0.0049), respectively. Cell survival
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*
4
2
*
Relative surviving fraction
Relative surviving fraction
30 Gy 3
2
1
0
2.5
5
7.5
10
15
Interval (min)
27
1
0.5
0
24
Dose (Gy)
Fig. 1. Relative surviving fractions of SCCVII tumor cells after 30 Gy given in 5 fractions of 6 Gy at intervals of 0–15 min and after 24 and 27 Gy as a single dose. Surviving fraction after continuous 30 Gy irradiation was regarded as 1. Bars represent SE of 6 determinations on average. *p < 0.05 against the control 30 Gy (0 min) group.
tended to increase with elongation of the interfraction interval, but not significantly. The hypoxic fraction for the control 1 cm SCCVII tumors was 18% (CI: 14–23%). Fig. 2 shows the results of the second experiment investigating reoxygenation after a priming dose of 13 Gy. The surviving fraction of the groups given second irradiation after cervical dislocation at intervals of 0 min was 0.00070 (CI: 0.00045–0.00094). No reoxygenation was seen when the interval was 0 or 2.5 min (hypoxic fraction: 100% at both intervals), but when the interruption was 5 min or longer, the hypoxic fraction was approximately 70% (67% at 5 min, 76% at 10 min and 66% at 15 min). The relative surviving fractions of the two groups were significantly different at 5 min after the priming dose 2.5
Relative surviving fraction
1.5
0 -2.5 0
2 1.5 1
2.5
5
7.5 10 Interval (min)
12.5
15
17.5
Fig. 3. Relative surviving fractions of SCCVII tumor cells after a priming dose of 5 Gy and a second dose of 15 Gy given at 0- to 15min intervals to air-breathing (s — s) or dead (d — d) mice. The surviving fraction of the group killed immediately after the priming irradiation was regarded as 1. Bars represent SE of 8 determinations on average.
(p = 0.047), indicating occurrence of significant reoxygenation. Fig. 3 shows the results of the third experiment investigating reoxygenation after a priming dose of 5 Gy. The surviving fraction of the groups given second irradiation after cervical dislocation at intervals of 0 min was 0.0081 (CI: 0.0038–0.012). Almost no reoxygenation appeared to occur within 5 min (hypoxic fraction: 83% at 0 min, 77% at 2.5 min and 81% at 5 min), but the hypoxic fraction tended to decrease to 57% (CI: 19–95%) at 10 min and 56% (CI: 31– 82%) at 15 min. Table 1 summarizes changes of hypoxic fractions at various intervals in the second and third experiments. Fig. 4 shows the results of the growth delay experiment. The effect of 25 Gy given in 5 fractions at 3-, 6- or 10-min interfraction intervals was significantly lower than that of a single 25 Gy dose (25 Gy as a single dose versus 25 Gy given in 5 fractions at 3-, 6- and 10-min intervals: p = 0.037, <0.0001, and <0.0001, respectively). The effect decreased or tended to decrease with elongation of the interfraction intervals (3-min intervals versus 6-min intervals, p = 0.045; 3-min intervals versus 10-min intervals, p = 0.002; 6-min
Table 1 Changes in the hypoxic fraction of SCC tumor cells at various intervals (0–15 min) after a priming dose of 13 or 5 Gy
0.5
Interval (min)
0 -2.5
371
0
2.5
5
7.5
10
12.5
15
17.5
Interval (min) Fig. 2. Relative surviving fractions of SCCVII tumor cells after a priming dose of 13 Gy and a second dose of 15 Gy given at 0- to 15min intervals to air-breathing (s — s) or dead (d — d) mice. The surviving fraction of the group killed immediately after the priming irradiation was regarded as 1. Bars represent SE of 8 determinations on average.
0 2.5 5 10 15
Hypoxic fraction (%) After 13 Gy
After 5 Gy
100 (61–100) 100 (64–100) 67 (41–93) 76 (34–100) 66 (37–100)
83 77 81 57 56
(68–99) (46–100) (52–100) (19–95) (31–82)
Figures in parenthesis are 95% confidence intervals (CI). The control SCCVII tumors had a hypoxic fraction of 18% (CI: 14– 23%).
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Biological effect of intermittent radiation
Relative tumor volume (%)
200
150
100
50
0 -10
0
10
20
30
40
50
60
Days after irradiation Fig. 4. Relative tumor volumes of SCCVII tumor cells after 20 or 25 Gy as a single dose, or 5 fractions of 5 Gy given at interfraction intervals of 3, 6 or 10 min. Tumor volumes before irradiation for each group were regarded as 100%. Bars represent SE of 18 mice on average. n, control; m, 20 Gy as a single dose; d, 25 Gy as a single dose; h, 25 Gy in 5 fractions given at 3-min intervals; j, 25 Gy in 5 fractions at 6-min intervals; s, 25 Gy in 5 fractions at 10-min intervals. Bars represent SE of 18 mice on average (for the irradiated groups).
Table 2 Tumor growth time (TGT) to reach 1.5 times the initial volume Radiation schedule
Control 20 Gy as a single dose 25 Gy as a single dose 25 Gy in 5 fractions at 3-min intervals 25 Gy in 5 fractions at 6-min intervals 25 Gy in 5 fractions at 10-min intervals
TGT (days) Mean
SE
3.2 39.5 55.1 52.4 50.5 46.9
1.4 5.1 3.1 3.9 3.8 4.6
intervals versus 10-min intervals, p = 0.24). Tumor growth time for each group is shown in Table 2.
Discussion In a previous study conducted by Shibamoto et al. [19] using 1-cm-diameter SCCVII tumors from the same frozen stock as used in this study, the hypoxic fraction was 5.4% (95% confidence interval [CI]: 4.0–7.4%) in air-breathing mice irradiated without anesthesia or physical restraint, but under the conditions for the growth delay assay (conditions for the fourth experiment in this study), the hypoxic fraction increased to 28% (CI: 20–40%). In other 3 studies by Shibamoto’s groups [11,18,20], the hypoxic fractions of the 1-cm-diameter SCCVII tumors from the same stock were 8.5% (CI: 6.9–11%), 10% (CI: 8–13%) and 9.1% (6.9–12%), respectively. Since immobilizing mice in jigs and taping their legs artificially increases the hypoxic fraction, oxygenation status is considered to remain poorer when tumor-
bearing mice are kept under these conditions [4,19]; in the fourth experiment (growth delay assay), reoxygenation was thus considered to be restricted compared with that in tumors in air-breathing mice with no physical restraint. Also from these observations, it was considered that mice had to be irradiated without physical restraint or anesthesia to investigate reoxygenation in the second and third experiments. The present study was undertaken in an attempt to find explanation for the discrepancy seen between our previous in vitro and in vivo studies; significant decreases in radiation effects were observed by posing intervals between fractions in vitro but they were not observed in vivo [16,17,21]. The apparent increase in cell survival in vitro should be due to SLDR, so questions arose as to whether SLDR might not occur in vivo or SLDR might be counterbalanced by other phenomena that sensitize tumor cells to subsequent irradiation. The first experiment was undertaken to investigate SLDR in vivo by excluding reoxygenation of tumor cells during fractionated irradiation. As a result, tumor cell survival tended to increase with elongation of intervals, but statistically, the increases were not significant. So, SLDR in vivo could not be proven with this experiment. One of the reasons why no statistically significant increases could be obtained in the first experiment may be the relatively large experimental errors associated with in vivo– in vitro studies. Moreover, clamping of tumor-bearing legs caused tumor bleedings, which led to a high frequency of bacterial contamination in vitro so that many experimental points were lost. This is also a reason why we used relative survival for analysis. This experiment was repeated 5 times (each time making 2 determinations per time point), so we did not further attempt to obtain statistical significance. The second and third experiments investigated the magnitude of reoxygenation during 2.5–15 min of radiation interruption. Several possible mechanisms for reoxygenation have been proposed, but rapid reoxygenation, if any, should mainly be due to restoration of blood flow to acutely hypoxic cells [1,13]. Kitakabu et al. [11] argued that the pattern of reoxygenation after a single 13 Gy dose seemed to be biphasic, comprising an extremely rapid phase within 1 h and a comparatively slow phase during the following 12– 72 h in SCCVII tumors. Reoxygenation within 1 h has also been reported in RIF-1 tumors [5,15], although it was hardly observed in EMT6 tumors [15,22]. Evidences showing that changes in blood perfusion can occur in subcutaneous SCCVII tumors within a 20-min interval were found in other studies [2,3]. Therefore, reoxygenation may occur even within 20 min in vivo. However, magnitude of reoxygenation within such a short time has not been well studied, so we attempted to clarify it in SCCVII tumors. In the present study, apparent reoxygenation was observed at 5 min after a priming dose of 13 Gy. Trends towards reoxygenation were also observed at 10 and 15 min, although the CI included 100%. After a priming dose of 5 Gy, both oxygenated and hypoxic cells can survive, but the hypoxic fraction tended to be lower at 10 and 15 min than at 0, 2.5 and 5 min. Taking these results together, reoxygenation appeared to take place within 15 min in SCCVII tumors. Then a question remains as to whether reoxygenation of this magnitude can counterbalance SLDR
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in vivo. In the second experiment, the hypoxic fraction was 100% at 0- and 2.5-min intervals, while it was 67% at 5 min interval, so approximately one-third of tumor cells reoxygenated within 5 min after 13 Gy. Surviving fractions for tumors irradiated in dead mice tended to increase with elongation of the intervals due to SLDR, but those for tumors irradiated in living mice remained at a level similar to that at 0 min, after 5-min or longer intervals following 13 Gy. In the third experiment, the hypoxic fraction was about 80% at 0- to 5-min intervals and about 55% at 10and 15-min intervals, so approximately one-third of tumor cells might also have reoxygenated at 10–15 min after 5 Gy. Surviving fractions for tumors irradiated in dead mice also tended to increase with elongation of the intervals due to SLDR, but those for tumors irradiated in living mice returned to a level similar to that at 0 min after 10- and 15min intervals. These result would indicate that reoxygenation of about one-third of hypoxic tumor cells would counterbalance SLDR in vivo. In the fourth experiment, the fractionated groups had faster tumor regrowth than the continuously-irradiated control group, and the effect of radiation tended to decrease with elongation of interfraction intervals. Thus, SLDR appeared to have occurred in this experiment, since no other explanations seemed relevant. As stated above, the hypoxic fraction in SCCVII tumors artificially increased from 5.4% to 28% by taping tumor-bearing legs [19]. In the present study, the control hypoxic fraction was 18% so that the hypoxic fraction of the taped and immobilized mice might have been somewhat higher. Anyway, the effects of intermittent exposure appeared to decrease, as compared with continuous irradiation, due to SLDR under these conditions. Results of the present study are summarized as follows. Two phenomena have been demonstrated in these tumor models that effect response in opposite ways during fractionated radiotherapy. The first was increased reoxygenation that can sensitize tumors (second and third experiments), and the second was presumably increased recovery (fourth experiment), although this was not formally proven in the first experiment. The reason why we could not prove SLDR in the first experiment was considered to be due to technical problems associated with leg clamping. In the fourth experiment, however, SLDR in vivo was considered to be proven by using the experimental setup which artificially restricted oxygenation. Potential relevance of our four series of experiments (Refs. [16,17,21] and the present study) for clinical radiation therapy may be summarized as follows. SLDR is a significant factor decreasing the effect of intermittent irradiation given over 20 min or longer. In some tumors in vivo, the decrease in effect may be compensated by rapid reoxygenation. However, the magnitude and velocity of reoxygenation in human tumors are almost unknown, so it cannot be concluded that the clinical effect of intermittent irradiation as employed in linac SRT is nearly equal to that of continuous irradiation. Biologically, investigations using tumors that are closer to human tumors are desirable. Clinically, the time for irradiation may better be kept as short as possible, considering the possibility of slow or insufficient reoxygenation of the tumor.
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Conflict of interest statement No conflicts of interest exist for any of the authors.
Acknowledgement This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Corresponding author. Natsuo Tomita, Department of Radiology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail address: [email protected] Received 25 March 2007; received in revised form 18 August 2007; accepted 20 August 2007; Available online 18 September 2007
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